Summary

An unconventional TGFβ superfamily pathway plays a crucial role in the
decision between dauer diapause and reproductive growth. We have studied the
daf-5 gene, which, along with the daf-3 Smad gene, is
antagonized by upstream receptors and receptor-regulated Smads. We show that
DAF-5 is a novel member of the Sno/Ski superfamily that binds to DAF-3 Smad,
suggesting that DAF-5, like Sno/Ski, is a regulator of transcription in a
TGFβ superfamily signaling pathway. However, we present evidence that
DAF-5 is an unconventional Sno/Ski protein, because DAF-5 acts as a co-factor,
rather than an antagonist, of a Smad protein. We show that expressing DAF-5 in
the nervous system rescues a daf-5 mutant, whereas muscle or
hypodermal expression does not. Previous work suggested that DAF-5 and DAF-3
function in pharyngeal muscle to regulate gene expression, but our analysis of
regulation of a pharynx specific promoter suggests otherwise. We present a
model in which DAF-5 and DAF-3 control the production or release of a hormone
from the nervous system by either regulating the expression of biosynthetic
genes or by altering the connectivity or the differentiated state of
neurons.

Introduction

Like many organisms, free-living nematodes can alter development in
response to environmental conditions. When food is scarce, population density
is high and temperature is high, animals arrest development after the second
molt as third larval stage (L3) dauers
(Golden and Riddle, 1984a;
Golden and Riddle, 1984b). Dauers are resistant to environmental insults and
do not age (Riddle and Albert,
1997), which allows survival and dispersal from environments with
poor resources. If conditions improve, the dauer molts and resumes
reproductive growth.

A transforming growth factor β superfamily (TGFβ) pathway is
required for a normal dauer decision, and is thought to act as a step in a
neuroendocrine pathway that couples external cues to dauer development
(Riddle and Albert, 1997;
Patterson and Padgett, 2000).
Food availability and population density are sensed as chemosensory cues.
These inputs, along with temperature, are sensed during early larval stages
(Golden and Riddle, 1984b). High food and low pheromone stimulate
transcription of daf-7, the gene for the ligand in the TGFβ
pathway (Ren et al., 1996;
Schackwitz et al., 1996).
DAF-7 binds to the receptors DAF-1 and DAF-4, which probably function in
neurons (Inoue and Thomas,
2000; Gunther et al.,
2000). DAF-8 and DAF-14 are Smads that appear to be the direct
targets of the receptors in the dauer TGFβ pathway (A. O. Z. Estevez, PhD
thesis, University of Missouri, 1997)
(Riddle and Albert, 1997;
Inoue and Thomas, 2000). These
Smads antagonize the function of another Smad called DAF-3
(Patterson et al., 1997). The
daf-5 gene has similar genetic properties to daf-3
(Thomas et al., 1993), and so
may be acting as a co-factor to DAF-3.

Little is known about events controlled by the receptors and Smads in this
pathway, or the mechanism by which the pathway controls the dauer decision.
Genetic analysis places the TGFβ pathway upstream of daf-12 (a
gene encoding a nuclear hormone receptor) and daf-9 (a gene encoding
a putative biosynthetic enzyme for a hormone that regulates DAF-12)
(Thomas et al., 1993;
Antebi et al., 1998;
Gerisch et al., 2001;
Jia et al., 2002). The
daf-9 gene appears to be expressed in the XXX cell, which is little
studied, but has neuronal properties and is located in a head ganglion
(Ohkura et al., 2003). These
facts, as well as the expression of DAF-7 in neurons and the suggested
function of DAF-4 in neurons (Inoue and
Thomas, 2000), suggest a model in which DAF-3 and DAF-5 function
in the nervous system. However, some evidence can be interpreted as suggesting
a function for DAF-3 and DAF-5 outside the nervous system. For example, a
small sequence element derived from a pharynx specific promoter binds DAF-3 in
vitro, and mediates daf-3-dependent repression of a reporter gene in
the pharynx (Thatcher et al.,
1999).

The dauer TGFβ pathway is unconventional in that upstream components
of the pathway directly or indirectly antagonize DAF-3, a Smad protein
(Patterson et al., 1997). In
other pathways, Smad transcription factors are activated, not repressed, by
the upstream components of the pathway. Anti-Smads antagonize receptor or Smad
function (Shi and Massague,
2003), but DAF-3 is antagonized by receptor signaling, and
functions in the absence of receptor signaling to regulate genes that control
dauer formation (Patterson et al.,
1997).

Genetic analysis is consistent with DAF-5 acting as a cofactor of the DAF-3
Smad. The most extensively studied roles of TGFβ superfamily pathways are
in the control of cell fate and in control of the cell cycle. The
neuroendocrine role of the dauer pathway is very different. The dauer
TGFβ pathway has evolved a unique mode of signaling, in which the
receptors and receptor-activated Smads antagonize another Smad protein, DAF-3.
Studies that reveal mechanisms of daf-5 function will help us
understand how signaling pathways evolve new functions.

We show that DAF-5 is a diverged homolog of human Sno and Ski, which
antagonize TGFβ signaling in cell culture by binding Smads, preventing
their interaction with each other and with co-activators, and by recruiting
co-repressors (Akiyoshi et al.,
1999; Luo et al.,
1999; Stroschein et al.,
1999; Sun et al.,
1999; Xu et al.,
2000; Frederick and Wang,
2002). DAF-5, Sno and Ski share a domain we call the SDS box; this
domain in Ski mediates its interaction with Smad4. Yeast two-hybrid
experiments demonstrate that DAF-5 interacts with DAF-3, and deletion studies
are consistent with this interaction being mediated by the same domains as in
the vertebrate Ski/Smad interaction. This functional similarity, combined with
our phylogenetic analysis of DAF-5/Sno/Ski homologs suggests that, despite a
highly divergent sequence, DAF-5 is an ortholog of Sno and Ski. DAF-5 is the
first example of a Sno/Ski with a genetically defined function in a TGFβ
pathway. We identified a mutation hotspot in the conserved Dachbox domain.
These mutants are the first direct evidence for a function for the Dachbox in
TGFβ signaling. Our analysis of regulation of a pharynx-specific promoter
does not support a previously hypothesized role for daf-3 and
daf-5 in the pharynx. Furthermore, we show that daf-5 is
expressed and functions in the nervous system. Therefore, we propose a model
in which daf-3 and daf-5 have evolved novel functions that
allow them to act in a neurosecretory pathway to control C. elegans
dauer developmental arrest.

Materials and methods

Phenotypic assays

Details of strains used can be found in supplemental data. For egg-laying
assays, worms were well fed and grown to L4 or young adult at 15°C, and
were transferred to continue development and lay eggs at 25°C. Animals
were scored 6 or 26 hours after the first egg was laid; for all genotypes, the
data were similar for scoring at any time in this interval. The total number
of eggs and the stage of the four oldest eggs inside of a worm were scored
with DIC optics. For dauer formation, synchronized worms were incubated at
25-25.8°C for 3-4 days, and scored for dauer formation. High temperature
Daf-c assays were very sensitive to temperature change; therefore, two
printing thermometers were used to record temperature every 2 hours. Reported
temperatures are an average of all readings. For scoring rnr-1 and
cki-1 expression, animals were grown at 15°C. Gravid adults were
transferred to fresh plates and allowed to lay eggs for 2-3 hours at room
temperature. The progeny were incubated at 23°C. Larvae were staged by
scoring the lethargus at the end of L2, and by body size and gonad size.

Mapping and transgenic strains

Orthology and paralogy

We use the terms ortholog and paralog as defined in
(Fitch, 1970) and
(Jensen, 2001). Simply put,
orthologs are duplicates of a character (e.g. a gene sequence) that arise from
speciation events, whereas paralogs arise from a duplication within a single
genome. Orthology and paralogy are typically hypotheses that are created based
on sequence comparisons and other data. These definitions make no suggestion
of conserved function.

Yeast-two-hybrid assays

Yeast two hybrid assays were performed as described
(Walhout and Vidal, 2001).
His/3-AT growth assay was scored on scale 0-3, and β-gal activity assay
was scored on scale 0-5. All protein-coding sequences of interest were fused
to the activation domain and the DNA-binding domain, and all assays were
performed with both AD/DB combinations. We assigned `+' scores based on the
total of His/3-AT and β-gal scores from the two combinations: ±,
1-3; +, 4-7; ++, 8 or 9; +++, 10-13; ++++, 14-16.

Results

DAF-5 is required for dauer formation and egg laying

We wished to evaluate the requirement for daf-5 in inducing dauer
formation when TGFβ pathway genes are mutant. Therefore, we carried out
an epistasis study of daf-5 using two alleles that are the most
likely nulls, based on gene structure (see below). Smads and receptors in this
pathway have redundant functions, and some single mutants do not completely
disable the pathway (Gunther et al.,
2000; Inoue and Thomas,
2000). In addition, the dauer TGFβ pathway is partially
redundant with a pathway that controls body size
(Krishna et al., 1999;
Morita et al., 1999).
Therefore, we tested for the ability of daf-5 mutants to suppress
when multiple pathway components are mutated. daf-8(sa343) and
daf-14(m77) have early stop codons, and each gives the strongest
known Daf-c (dauer formation constitutive) phenotype for the respective genes
(A. O. Z. Estevez, PhD thesis, University of Missouri, 1997)
(Inoue and Thomas, 2000). At
25°C, we see complete suppression of the Daf-c phenotype of daf-8;
daf-14 double mutants and daf-8; daf-14; sma-2 triple mutants
(Table 1). Therefore, at this
temperature, daf-5 is essential for dauer formation induced by
TGFβ pathway mutants. At slightly higher temperatures, suppression is
incomplete, and continues to diminish as the temperature is raised from
25.4°C to 25.8°C. Our results are consistent with previous results
(Thomas et al., 1993;
Ailion and Thomas, 2000), but
we show that failure to completely suppress daf-c mutants at
temperatures higher than 25°C is not caused by incomplete loss of
daf-5 function (previous studies used alleles of daf-5 that
may not be null).

Animals mutant for daf-8 or daf-14 Smad genes also have
an Egl-d (egg laying defective) phenotype. These animals have a structurally
normal reproductive system, but lay eggs less frequently than wild type, and
accumulate eggs inside. This phenotype is suppressed by daf-5 and
daf-3 mutations (Trent et al.,
1983; Thomas et al.,
1993). daf-8, daf-14 and daf-8; daf-14 mutants
contained more eggs (Table 2)
and older eggs (see Table S1 and Fig. S1 at
http://dev.biologists.org/supplemental),
than wild type. Introducing daf-5 mutants into daf-8 or
daf-14 mutants or daf-8; daf-14 double mutants fully
suppressed the Egl-d phenotype, except in the case of daf-8; daf-5(mg89);
daf-14, which was intermediate between wild type and the Daf-c mutants.
Interestingly, daf-5 single mutants have fewer eggs in the uterus
than the other genotypes, and have younger eggs inside (for example, 50% of
the oldest eggs in daf-5(sa310) have fewer than eight cells, versus
15% in wild type). This Egl-c (egg-laying constitutive) phenotype has not been
previously reported for daf-5 mutants.

Cloning of daf-5

We identified the daf-5 coding region using DNA polymorphism
mapping, and transgenic rescue of mutants. Our three factor mapping (data
submitted to WormBase) placed daf-5 in a ∼120 kb interval
(Fig. 1A). Cosmid rescue of
daf-5 mutants suggested that the daf-5 gene was contained on
the cosmid W01G7. Only one predicted gene was within the interval to which
daf-5 was mapped (Fig.
1A). We sequenced three cDNAs provided by Y. Kohara. The longest
clone, yk130g8, is identical to the structure inferred from a
concatenation of EST sequences shown by WormBase
(http://www.wormbase.org)
and GenBank (NM_064540).

DAF-5 encodes a C. elegans homolog of the Sno and Ski
oncoproteins. (A) Identification of daf-5-coding region. The genetic
map near daf-5 and rescued transgenic lines isolated are shown. (B)
DAF-5 gene structure. At the C terminus of DAF-5, a stop codon and a deletion
mutant are connected by a broken line to indicate that these two mutations
were identified in a single allele. A mutation hotspot is shown; an additional
mutant has an in-frame deletion of the 15 amino acids shown. Colors of amino
acids in hotspot represent sequence conservation as in Fig. 1C,D. See Table S2
at
http://dev.biologists.org/supplemental
for complete details on mutant alleles. (C,D) Alignment of SDS and Dach boxes.
Consensus at each position was defined as any set of identical and similar
amino acids that were found in more than one subgroup (subgroups are indicated
by spaces between the rows of sequence), and in more than three proteins in
total. Bold magenta residues are identical in the primary consensus (the
consensus with the most matches) and plain, magenta are similar. Green
residues are the secondary consensus. The % symbols below the SDS box show
conserved zinc chelating residues, and the asterisks indicate amino acids that
contact Smad4 in the crystal structure of a Ski/Smad4 complex. The sequence
for Dog Nem. is from an EST, and has two stop codons in frame (indicated by
X). These may result from sequencing errors, or the cDNA may have come from a
pseudogene. Accession Numbers and abbreviations: Dros., D.
melanogaster (Iceskate: NP_651946; Snowski: NP_609166; Dachshund:
NP_723972); Mosq., mosquito (A. gambiae; Iceskate-XP_317739;
Snowski-XP_317545); Soy. Nem., Soybean Cyst nematode (Heterodera
glycines; CA939358); Root nem., Root Knot nematode (Meloidogyne
chitwoodi; CB931358); Pot. Nem., Potato Cyst nematode (Globodera
rostochiensis; AY389814); Lymph Nem, Brugia malayi (AY389813);
Dog Nem, dog hookworm (Ancylostoma caninum; AW735310); C.
briggsae DAF-5 (found in Wormbase as CBG20832); C. elegans
(DAF-5-NM_064540, NP_496941; Dachshund-NP_497266); Human (Dachshund-NP_542937;
cSki-NP_003027; SnoN-NP_005405; Icy-XP_292349; Skate-XP_064560); sea squirt
(Ciona intestinalis; Ski-BK001616; Dachshund-AABS01000073).

DAF-5 is similar to Ski

A blast of the predicted DAF-5 protein sequence against databases at NCBI
reveals similarity to the oncoproteins Ski (for Sloan Kettering Virus) and Sno
(for Ski-related novel sequence). A careful examination of many Sno/Ski
homologs demonstrates that the homology of DAF-5 is significant, and we
suggest that DAF-5 is the C. elegans ortholog of Sno/Ski. We show two
domains from the Sno/Ski/Dachshund superfamily.
Fig. 1C shows the SDS box (for
Sno, Daf-5 and Ski); in human Ski, the SDS box and about 20 amino acids on
either side constitute the minimal region required for binding to Smad4.
Fig. 1D shows the Dachbox-N, a
domain shared by the Sno/Ski family and Dachshund, which is a transcription
regulator conserved throughout bilateria. DAF-5 has a predicted coiled-coil at
the C terminus; Sno and Ski also have a coiled coil, but have a pattern of
charged residues not shared by DAF-5.

This alignment is very informative regarding the relationship of DAF-5 to
other members of the family. Sno and Ski are found in humans and all major
groups of vertebrates, but in insects have only one ortholog of these two
proteins (see Materials and methods for definitions of ortholog and paralog).
Sno and Ski are more similar to each other than either is to the single
Drosophila or mosquito ortholog; therefore Sno and Ski are probably
paralogs that were duplicated after the divergence of the protostome and
deuterostome lineages. We have named the insect genes Snowski
(Snk) to reflect the orthology to both Sno and
Ski.

A new family of proteins closely related to the Snowski group is shown in
Fig. 1C,D. Humans have two
genes in this group. We have named these genes Skate (for
Ski-related gene) and Icy (for Ski sequence
family). Drosophila and mosquito each have a gene that is much
more similar to human Icy and Skate than to Drosophila
Snk. We have named the single Drosophila and mosquito genes
iceskate (isk) to reflect their orthology to both
Icy and Skate.

We suggest that DAF-5 is an ortholog of Snowski or Iceskate. First, DAF-5
clearly has an SDS box, which is not found in any other protein in C.
elegans or C. briggsae. This SDS box is more similar to the
Snowski group than to the Iceskate group, including amino acids that are
important for the ability of Ski to bind Smad proteins. Second, DAF-5 binds
the DAF-3 Smad (see below). This binding is mediated by the SDS box in Ski,
and may thus be a conserved function of the SDS box. Third, the rate of
divergence in the Snowski/Iceskate family is so high that relatively modest
sequence conservation is not surprising. This rapid change can be seen when
examining the SDS box of B. malayi and potato cyst nematode Snowski.
These two nematode proteins are more different from each other than insect
Snowski is from Human Sno and Ski. The DAF-5 gene is even more rapidly
diverging in the Caenorhabditis genus. C. briggsae and
C. elegans proteins average more than 70% amino acid identity. The
DAF-5 sequence is only 40% identical overall between C. briggsae and
C. elegans. In fact, in the Dachbox and SDS box, the difference
between C. elegans and C. briggsae DAF-5 is greater than the
difference between insect and human Snowski.

A mutation cluster in the Dachbox domain

Sequencing of daf-5 mutants identified a mutation hotspot. We
identified mutations in 15 daf-5 alleles
(Fig. 1B; see Table S2 at
http://dev.biologists.org/supplemental).
All five missense mutants were found in a 16 amino acid stretch of the 627
amino acid protein. This hotspot is in the region of the Dachbox where DAF-5
is most similar to Snowski, Iceskate and Dachshund. Three of the mutants
affect two very strongly conserved residues (two of the mutants are identical
but independently isolated). One mutant has an in frame deletion of this
region, and the final mutant is in a glycine that is unique to DAF-5. This
region is critically important for DAF-5 function; the sa310 mutation
(E162K) has a phenotype as severe as putative null alleles
(Table 1). In vitro analysis
showed that an insertion of four amino acids next to residue 168, which is
homologous to the residue that is mutated in daf-5(sa310), eliminates
the transforming and myogenic activity of vSki
(Zheng et al., 1997). Thus,
the Dachbox is required for a gain-of-function phenotype of vSki, but how this
function relates to wild-type function of cSki or to TGFβ signaling has
not been experimentally determined. A point mutation in the Dachbox disrupts
the interaction of Ski with NCoR (Ueki and
Hayman, 2003). However, this mutation affected Vitamin D
receptor-dependent gene expression, but not TGFβ-dependent gene
expression. These in vitro experiments suggest possible functions for the
Dachbox, but our daf-5 mutants are the first missense mutants
identified in any Sno/Ski gene, and thus the first in vivo evidence
for the wild-type function of a particular domain of Sno/Ski.

DAF-5 binds to Smad DAF-3

A yeast two-hybrid screen for proteins that interact with DAF-3 identified
the predicted protein W01G7.1 (M. Tewari, P. J. Hu, G. B. Ruvkun and M. Vidal,
personal communication), which we show is DAF-5. We used yeast two-hybrid
assays to identify regions of DAF-3 and DAF-5 required for interaction. We
find that the DAF-3 MH2 domain strongly interacts with a DAF-5 fragment that
is truncated after the SDS domain (Fig.
2, see Table S3 at
http://dev.biologists.org/supplemental).
Thus, as in vertebrates, the region downstream of the SDS box is dispensable
for binding to Smads and the Smad domain that binds Sno/Ski is the MH2 domain
(Akiyoshi et al., 1999;
Luo et al., 1999;
Stroschein et al., 1999;
Sun et al., 1999;
Frederick and Wang, 2002;
Wu et al., 2002). These
interaction results suggest that DAF-3 and DAF-5 function as part of a
transcriptional regulatory complex.

Binding of DAF-3 to DAF-5 in yeast two-hybrid assays. Interactions were
scored by transcriptional activation of a His reporter and a β-gal
reporter. The strength of activation was assigned a score in each assay, and
the symbols reflect the sum of the scores for all assays (see Materials and
methods).

DAF-5 is found in nuclei of neurons, pharynx and some other
tissues

We made reporter gene GFP fusions to identify the cells in which
daf-5 might function. Our full-length construct rescues a
daf-5 mutant as efficiently as a wild-type genomic clone
(Table 4). We saw relatively
strong expression in ganglia in the head and tail and in the anterior pharynx
(Fig. 3). Cell ablation and
other experiments have shown that several cells in the nervous system (ASI,
ADF, ASG, ASJ and XXX) are required for normal regulation of dauer formation
(Bargmann and Horvitz, 1991;
Schackwitz et al., 1996;
Jia et al., 2002;
Gerisch et al., 2001). We
examined these cells for DAF-5::GFP expression. GFP was not seen in ASI, ASJ,
ADF or ASG (0 of 86 animals). Fluorescence in the anterior ganglion, where XXX
is found, is at least an order of magnitude less than in the bright cells of
the ganglia posterior to the nerve ring. The weakness of fluorescence has
precluded identification of specific cells, but we sometimes see weak
fluorescence in the ventral, anterior part of the ganglion, where XXX resides
(seven out of 53 animals). A small number of animals show weak expression in
the hypodermis, muscles, intestine and distal tip cells (see Table S4 at
http://dev.biologists.org/supplemental).
We also constructed a transcriptional GFP fusion, which consists of 6.5 kb
upstream of the first ATG and also the first 57 codons in the first exon. The
non-rescuing GFP construct is more strongly expressed, and shows more
consistent expression in the hypodermis, muscles, intestine and distal tip
cells; still, its expression is strongest in the head and tail ganglia (see
Table S4 at
http://dev.biologists.org/supplemental).
DAF-5::GFP from the rescuing construct mostly localizes to nuclei
(Fig. 3,
Table 3), which is consistent
with the idea that DAF-5 is a transcription factor and functions in the
nucleus. We examined the intensity of fluorescence and nuclear localization of
DAF-5::GFP from the rescuing construct in wild-type and in TGFβ pathway
mutants, including dauers, and saw no obvious differences.

daf-5 is expressed in the nervous system and preferentially
localized to nuclei. Expression of functional daf-5::GFP. (A)
Functional daf-5::GFP is predominantly expressed in head ganglia.
Several neurons in the head ganglia are indicated by arrowheads. (B) DAF-5 is
localized to the nucleus. This panel is an enlargement of the area boxed in A.
A triangle shaped neuron was glowing with DAF-5::GFP mostly found in its oval
nucleus.

daf-5 functions in nervous system

We used tissue-specific expression of daf-5 to identify cells in
which it functions. We expressed daf-5cDNA::GFP with various
tissue-specific promoters (Aamodt et al.,
1991; Okkema et al.,
1993; Hsu et al.,
1995; Maduro and Pilgrim,
1995; Gilleard et al.,
1997; Ogura et al.,
1997); these constructs had GFP inserted at the same site as a
genomic construct that rescued a daf-5 mutant
(Table 4). Rescue was assayed
in daf-7; daf-5 double mutants. Rescued animals would be expected to
have the Daf-c phenotype of a daf-7 mutant. pF25B3.3
strongly expressed daf-5::GFP exclusively in nervous system and
rescued daf-5 mutants as well as two positive controls. Similarly,
punc-14, which expressed daf-5::GFP in nervous system at
high level in addition to some non-neuronal expression, also showed strong
rescue. Weak ubiquitous expression of daf-5::GFP from the
pdpy-30 promoter gave partial rescue. unc-119::daf-5::GFP
was weakly expressed in the nervous system but did not rescue, perhaps owing
to the low level of expression. Strong expression of daf-5::GFP from
the muscle promoter pmyo-3 did not rescue at all. Initial tests of
two strains of pdpy-7::daf-5::GFP gave very weak expression and no
rescue. Therefore, we isolated additional lines that had strong hypodermal
expression of daf-5::GFP, and these did not rescue either. For
unknown reasons, the expression of pges-1::daf-5::GFP was
undetectable. Overall, our results show neuronal expression of daf-5
is sufficient to rescue daf-5 dauer formation defect, while muscle or
hypodermal expression is not.

DAF-5 is required for cell cycle arrest

In vertebrate cells, Smad proteins control the cell cycle by regulating
transcription of cyclin kinase inhibitor genes
(Moustakas and Kardassis,
1998). The division of hypodermal seam cells is arrested in dauers
because CKI-1, a cyclin kinase inhibitor, is transcriptionally upregulated,
and this upregulation is inhibited by wild-type daf-7
(Hong et al., 1998),
suggesting that daf-5 may directly or indirectly upregulate
cki-1. We tested whether cell cycle arrest in dauers requires
daf-5. The reporter, rnr-1::GFP, drives the expression of
GFP in the seam cells as they enter S phase and through the division at the
beginning of L3. daf-7 (e1372) mutants do not express
rnr-1::GFP at the corresponding time, which reflects seam cell cycle
arrest in dauers (Hong et al.,
1998). We observed that 47% of N2 show rnr-1::GFP
expression (Fig. 4A,
Table 5), whereas none of the
daf-1(sa184)-induced dauers does. Thus, this daf-1
mutation causes a complete arrest of the cell cycle. Interestingly, in
daf-5(sa310); daf-1(sa184) double mutants, the
percentage of animals with green seam cells is 31%, which is similar to wild
type (Fig. 4B;
Table 5). The reason that we
did not see rnr-1 expression in some N2 or daf-5; daf-7
double mutants even after careful synchronization was probably due to the
transient expression of the rnr-1 gene and variation in the timing of
cell cycle among individual animals. We conclude that a daf-5
mutation suppresses the cell cycle arrest caused by daf-1.

daf-5 controls cell cycle arrest in the seam cells of dauer larvae
by regulating expression of a cyclin kinase inhibitor. (A,B)
rnr-1::GFP expression in N2 and daf-5; daf-1 double mutants.
A corresponding Nomarski image can be found in Fig. S2 at
http://dev.biologists.org/supplemental.
White arrowheads show seam cells that express rnr-1::GFP in N2 and in
daf-5; daf-1, respectively, at early L3 stage. (C,D)
cki-1::GFP expression in daf-1 and daf-5; daf-1
mutants. A corresponding Nomarski image can be found in Fig. S2 at
http://dev.biologists.org/supplemental.
(C) cki-1 is expressed along the seam cells in all daf-1
dauer larvae with an average of 16 green cells. White arrowheads (E) show seam
cell nuclei. (D) cki-1 is expressed in a subset of daf-5;
daf-1 mutants in early L3. Two daf-5; daf-1 animals are shown.
The top animal is representative of the 37% of animals of this genotype that
had no GFP in the seam at all. The bottom animal is representative of the 34%
that expressed GFP faintly in many seam cells (between five and ten). White
arrows point to the seam cells that do not express cki-1::GFP in the
nuclei. Scale bars: 1 μm.

We wanted to test whether daf-5 controls seam cell cycle arrest
via the cyclin kinase inhibitor, cki-1. In wild type, after cell
division is complete, cki-1::GFP expression is seen, reflecting the
role of cki-1 in restricting seam cells to one and only one division
(Hong et al., 1998). In dauer
larvae, cki-1 is expressed during the period where seam cells would
divide in wild type; as a result, the seam cells do not divide. In
daf-1(sa184) induced dauers, 100% of the animals with the
cki-1::GFP reporter had a continuous line of green seam cells on both
sides (Fig. 4C,
Table 5), confirming the
regulation of cki-1 by TGFβ signaling
(Hong et al., 1998).
Eighty-one percent of wild-type animals had either no green seam cells (53%)
or a few faintly green seam cells (28%) expressing GFP. Similar to the N2
animals, 66% of daf-1; daf-5 double mutants had either no green seam
cells (37%) (Fig. 4D, top
animal, Table 5) or a few
faintly green seam cells (29%). Nineteen percent of N2 and 34% of daf-1;
daf-5 double mutants have many green cells
(Fig. 4D bottom animal,
Table 5). Expression in a
subset of animals is expected because cki-1 is transiently expressed
at the beginning of each larval stage to limit seam cells to a single division
(Hong et al., 1998).
Therefore, daf-5 mediates cell cycle arrest in dauers by controlling
cki-1 expression. However, unlike in vertebrate TGFβ signaling,
this control is likely to be indirect, because daf-5 expression in
the nervous system is sufficient to cause dauer arrest.

Regulation of myo-2

Previous work is consistent with DAF-5 and DAF-3 having a function in the
adult pharynx, but we found that expression of DAF-5 solely in the nervous
system can cause dauer arrest. Therefore, we re-examined the role of DAF-3 and
DAF-5 in adult pharynx. DAF-3 was shown to bind to a 5 bp sequence within a 32
base pair regulatory element isolated from the C. elegans myo-2
promoter (the `C subelement'). This regulatory element drives pharynx-specific
gene expression when placed upstream of a minimal promoter
(Thatcher et al., 1999).
Expression from this construct in adults is strongly repressed in mutants of
daf-7 and other Daf-c mutants in the TGFβ pathway, but a
mutation of daf-3 or daf-5 relieves this repression.
However, all of this regulation was observed with the C subelement removed
from its normal context and concatamerized upstream of a minimal promoter, and
we wished to see if this regulation occurs within the normal context of the
full myo-2 promoter.

We used a GFP reporter fused to a full-length myo-2 promoter (with
1.6 kb upstream of the translation start) to examine regulation by genes in
the TGFβ pathway. If the full-length promoter is regulated similarly to
the C subelement reporter, we expected that expression would be repressed in
daf-7 mutant adults relative to wild-type adults, and that mutations
in daf-5 or daf-3 would alleviate that repression. However,
expression in adults was indistinguishable in wild type and in daf-7
mutants (Table 6). Therefore,
the regulation of the full-length promoter is unlike the regulation of the C
subelement reporter. Similarly, in wild-type L2 stage larvae and
daf-7 larvae in the corresponding L2d stage, expression was
indistinguishable between N2 and daf-7, and expression in the
daf-7;daf-5 and daf-7;daf-3 double mutants was modestly
reduced. Finally, we see that expression in the daf-7 dauer is less
than in wild type or the daf-7;daf-3 and daf-7;daf-5 double
mutants. The C subelement reporter also shows a reduction in expression in
dauers, comparable with what we see. However, unlike in adults, the reduction
of expression of the C subelement reporter in dauers is not dependent on
daf-3 (Thatcher et al.,
1999), and is therefore mediated by some other pathway. In
summary, we see no evidence that the full-length myo-2 promoter is
regulated by daf-3 or daf-5.

Discussion

We show that DAF-5 is homologous to the Sno/Ski family of transcriptional
co-repressors, and we argue that DAF-5 is likely to be the C. elegans
ortholog of human Sno/Ski and Drosophila Snowski. In vertebrates, Sno
and Ski antagonize TGFβ signaling by binding to Smads, but our analysis
clearly demonstrates that DAF-5 is not an antagonist of the DAF-3 Smad. We
show that DAF-5 is localized to the nucleus, and that it binds DAF-3, which
strongly suggests that DAF-5 is a co-factor of DAF-3, and that DAF-5 assists
DAF-3 in regulating gene expression. Our findings are novel in that we have
identified a wild-type function for a Ski/Sno protein in a TGFβ pathway
in vivo.

Analysis of DAF-5 identifies a role for a Snowski family protein in
TGFβ signaling in vivo

Recent work in cell culture has showed that Sno and Ski function in vitro
as key factors in TGFβ regulation of gene expression and cell division
(Akiyoshi et al., 1999;
Luo et al., 1999;
Stroschein et al., 1999;
Sun et al., 1999;
Wang et al., 2000;
Xu et al., 2000;
Frederick and Wang, 2002;
Miyazono et al., 2003;
Ueki and Hayman, 2003). These
studies suggest numerous possible functions for Sno and Ski in TGFβ
signaling, but understanding the specific contexts in which Sno and Ski
function in vivo will require further work. Some studies of Sno/Ski function
in vivo have used gain-of-function phenotypes in Xenopus development
(Wang et al., 2000) or in
cancer (Frederick and Wang,
2002; Miyazono et al.,
2003). These studies have identified mechanisms of Sno/Ski action
and suggest possible roles for the proteins in vivo, but are accompanied by a
caveat. Overexpression and gain-of-function mutants might cause the genes to
act in an event that is not part of the normal function of the gene. Mouse
Ski and Sno mutants have interesting phenotypes, including
skeletal and muscular developmental defects and cancer susceptibility
(Berk et al., 1997;
Shinagawa et al., 2000;
Shinagawa et al., 2001). These
mutants will be useful in dissecting the wild-type functions of Sno and Ski in
TGFβ signaling; however, the phenotypes of these mutants are complex, and
Sno and Ski function in multiple signaling pathways
(Dahl et al., 1998;
Nomura et al., 1999), so tying
a specific phenotype to the effect of Sno or Ski on TGFβ signaling will
require careful analysis. Whereas Sno and Ski function in many pathways,
daf-5 mutants exclusively affect phenotypes controlled by the dauer
TGFβ pathway; this fact makes the assignment of function much more
straightforward in C. elegans.

DAF-5 and the Snowski/Iceskate gene families

From analysis of the Snowski family, we identified several interesting new
genes and relationships among them. First, we show that Sno and
Ski are likely to be paralogs that arose after the divergence of
protostomes and deuterostomes, perhaps as late as the divergence of
urochordates and cephalochordates (because sea squirt has only one ortholog).
Second, we identify the Iceskate family: an uncharacterized, highly
conserved Snowski-related gene family. Genes in the Iceskate
family have a striking difference from Snowski. Iceskate shows
virtually no similarity to a set of amino acids in the SDS box of Ski that
provide critical hydrogen bonds and van der Waals contacts in the Ski/Smad4
structure (Wu et al., 2002).
To our knowledge, Iceskate proteins have not been studied experimentally; the
functional relationship of Iceskate to Sno/Ski is an interesting issue for
future research. Third, we find that Snowski is changing rapidly in nematode
evolution. In Caenorhabditis, we see that daf-5 is changing
rapidly, much more rapidly than the typical C. elegans gene, which
may explain the relatively low primary sequence conservation between DAF-5 and
other Snowski proteins.

Despite the rapid evolution of DAF-5, it binds DAF-3, a Smad. We find that
the region downstream of the SDS box is entirely dispensable for DAF-5 binding
to Smads, and the MH2 domain of DAF-3 is sufficient for interaction with
DAF-5. The region of DAF-5 homologous to the region of Ski that contacts Smad4
is shown in Fig. 1C. Sno, Ski,
DAF-5 and all other members of this family have a conserved zinc finger in the
SDS domain. This zinc finger is an important structural component of Ski, and
presumably all members of this family, because it is absolutely conserved.
Only a subset of the residues of Ski that contact Smad4 are conserved in
DAF-5. The binding of DAF-5 to DAF-3 may be somewhat different from the
binding of Ski to Smad4, because DAF-3 is also greatly diverged from
Smad4.

Evolution of the dauer TGFβ pathway

The rapid change in daf-5 in the Caenorhabditis genus
prompts the question of to what extent have daf-5 and other members
of the TGFβ pathway evolved new functions? The model for Sno/Ski function
in vertebrate TGFβ signaling is shown in
Fig. 5A. Briefly, activation of
TGFβ receptors causes phosphorylation of Smads. This activation allows
the Smads to bind Sno/Ski proteins and cause their degradation. With Sno/Ski
gone, the Smads are free to regulate gene expression. However, Sno
and Ski transcription is activated by Smads, leading to accumulation
of Sno/Ski protein after receptor activation. Sno and Ski bind to Smads and
recruit a variety of co-repressors, which prevent Smads from activating gene
expression.

Model for Sno/Ski and DAF-5 function. (A) Antagonism by Sno and Ski in
vertebrates. (B) Function of DAF-5 in C. elegans. See Discussion for
explanation of models.

Two major functional changes have occurred within the dauer TGFβ
pathway (Fig. 5B). First, DAF-3
is unique among Smads in that its function is antagonized by receptor
signaling, and that it acts in the absence of the receptors and R-Smads. DAF-3
might recruit factors that in other systems are recruited by R-Smads, or the
pathway may have evolved to use other factors. Second, DAF-5 is the only known
example of a Snowski protein acting in a TGFβ pathway as a co-factor
rather than an antagonist. In principle, Sno or Ski could act as cofactors
rather than antagonists on promoters that are negatively regulated by
TGFβ, but this function has not yet been observed. One function that
DAF-5 probably does not retain is a function in a variety of signal
transduction pathways. Ski acts as a co-repressor for Mad, multiple nuclear
hormone receptors, Rb, and other transcriptional regulators
(Nomura et al., 1999;
Dahl et al., 1998);
Sno or Ski mutant mice have severe developmental defects,
consistent with their role in multiple pathways
(Berk et al., 1997;
Shinagawa et al., 2001).
daf-5 mutants, however, have much more modest defects, and
daf-5 has no known phenotypes that are not shared by daf-3,
suggesting that DAF-5 acts only as a co-factor to DAF-3.

DAF-5 acts in the nervous system

The question of where the TGFβ pathway is acting is important, and
evidence is not conclusive. Many of the genes in the pathway have broad
patterns of expression, including many non-neuronal cells
(Patterson et al., 1997;
Gunther et al., 2000;
Inoue and Thomas, 2000). We
found that expression from a neuron-specific promoter efficiently rescued the
daf-5 mutant, and that expression in muscle or hypodermis did not
rescue. This result is consistent with daf-5 acting in neurons to
regulate a hormonal cue for dauer.

Experiments to directly address the site of action of DAF-4 have been
reported (Inoue and Thomas,
2000), and suggest that this gene also has a neuronal focus of
action. In these experiments, a daf-4 cDNA was fused to several
tissue-specific reporters. A promoter expected to give neuronal and intestinal
expression fused to a daf-4 cDNA rescued a daf-4 mutant, but
putative intestine-specific, muscle-specific and other promoters did not.
However, the authors could not monitor the expression of the constructs
directly, and they were appropriately cautious in interpreting their results.
Our results suggest that unexpected expression is not just a formal
possibility. Rather, misexpression of `tissue specific' reporter constructs
may be common. Several of our constructs gave unexpected expression patterns
(Table 4). The unc-14
promoter has been reported to be neuron-specific, but we saw expression
elsewhere. The unc-119, dpy-7, dpy-30 and ges-1 promoters
gave unexpectedly weak or undetectable expression, although in the case of
dpy-7, we were able to correct the problem by isolating transgenic
strains with stronger expression.

Two reports suggest functions for daf-3 and daf-5 outside
the nervous system, which is inconsistent with our conclusion. One report
suggested that daf-3 and daf-5 function in the pharynx to
directly regulate gene expression in adults
(Thatcher et al., 1999). This
work used a reporter with a small element derived from the myo-2
reporter (the `C subelement'). However, our examination of the full-length
myo-2 promoter indicates that daf-3 and daf-5 do
not regulate myo-2 gene expression in adults. In dauers, the
full-length promoter does show regulation similar to that of a reporter
containing only the C subelement, but regulation of the C subelement reporter
in dauers is daf-3-independent. This daf-3-independent
downregulation may be caused by a general reduction in expression of
housekeeping genes that is seen in dauers (T. Liu and G.I.P., unpublished). We
suggest that daf-3 and daf-5 do not actually regulate the
expression of myo-2 in a normal context. daf-3 and
daf-5 may function in the nervous system to regulate a secondary
signal that in turn regulates the reporter with the C subelement.

Cell division arrest in hypodermis and other non-neuronal cells of dauers
is dependent on a cyclin kinase inhibitor, cki-1
(Hong et al., 1998). This gene
is similar to a gene that is directly regulated by Smads in vertebrates
(Moustakas and Kardassis,
1998). In C. elegans, this gene is repressed in a
daf-7 mutant, and we found that this regulation was daf-5
dependent, which suggests that daf-3 and daf-5 could act
outside the nervous system to directly regulate this gene. However, our
demonstration that daf-5 expression in the nervous system is
sufficient for dauer arrest suggests that regulation of cki-1 is
indirect.

Biological role of TGFβ signaling in C. elegans

We observed a phenotype of daf-5 that has not been previously
reported; daf-5 mutants are Egl-c, meaning that compared with wild
type, daf-5 mutants have fewer eggs retained in the uterus and lay
eggs with younger embryos. This phenotype suggests that the daf-5
mutants have hyperactive egg-laying behavior. We were concerned that a low
rate of egg production in the spermatheca might cause this phenotype, but we
found that the rate of egg production was not correlated with the Egl
phenotype. We found that all of the mutant genotypes in our experiment produce
eggs at a rate of 1/2 to 2/3 that of wild type (data not shown), but have
dramatically different Egl phenotypes. For example, the daf-5 single
mutants make eggs at the same rate as the daf-8; daf-5; daf-14 triple
mutants, which are not Egl-c. This new result suggests that daf-5
functions to restrain egg laying even when the TGFβ pathway is active.
The role of daf-5 in egg laying is not TGFβ pathway independent,
as daf-c; daf-5 double mutants are not significantly Egl-c.

In addition to dauer and egg laying, the dauer TGFβ pathway also
regulates social feeding behavior and fat accumulation
(Trent et al., 1983;
Thomas et al., 1993). In
addition, daf-3 and daf-5, but not the other Smads or
receptors, have a role in chemotaxis to both olfactory and gustatory cues
(Daniels et al., 2000). All of
these behaviors and developmental events are also affected by chemosensory
input. Thus, the role of the TGFβ pathway can be said to be a general
role in coupling chemosensory events to developmental and behavioral output.
How might this pathway be coupled to these events? One possibility is that all
of these events are affected, directly or indirectly, by a hormonal cue or
cues, and that the TGFβ pathway regulates the expression of genes needed
for production of the hormone(s). Orientation to a chemical gradient is quick,
occurring within minutes. This is perhaps too brief an interval for the
hormone to be acting during the chemotactic process, but a hormone might cause
structural changes in the nervous system that alter chemotactic behavior.

A second interesting possibility is suggested by recent work that has
identified new functions for TGFβ superfamily signaling in the nervous
system. Retrograde signals between Drosophila neurons and their
postsynaptic partner cells can affect the nature of the synaptic connection
(Aberle et al., 2002;
Marques et al., 2002) as well
as the neurotransmitters produced by the presynaptic cells
(Allan et al., 2003), and
genetic analysis suggests that these retrograde signals are mediated by Gbb, a
TGFβ superfamily ligand, and Wit, a TGFβ superfamily receptor. Thus,
the dauer TGFβ pathway might act to affect the synaptic connections or
other properties of neurons to alter signaling in the nervous system. This
model is not mutually exclusive with the hormone regulation model above, as
changes in connectivity or neurotransmitter production might affect synaptic
signaling to a neurosecretory cell that makes hormone.

Gene regulation by the Ski superfamily

Study of the Ski and Sno proteins has provided an opportunity to learn how
a co-repressor can play a variety of roles in different regulatory events.
Perhaps the next important task will be to tie the biochemical and cell
biological mechanisms identified in cell culture systems to important
functions of Sno and Ski in vivo. Cancer biology is one field that may provide
this connection, as the role of Ski and Sno in cancerous cells suggests
testable hypotheses about the role of these proteins in the normal context.
Sno and Ski function in multiple pathways, which makes understanding the role
of the proteins in any one pathway difficult. In C. elegans, DAF-5
appears to function predominantly or exclusively in a single TGFβ
pathway. This simplicity has allowed us the unique discovery of an unambiguous
connection of a Sno/Ski family member to a specific regulatory event in vivo,
and this discovery provides a genetic system in which to pursue further
understanding of Sno/Ski function.

Acknowledgments

We thank Dr Cole Zimmerman and Dr Richard W. Padgett for
rnr-1::GFP construct; Dr Victor Ambros for strain VT825; J. Babiarz
for microinjection; L. Tarantino for construction of daf-5 mutant
strains; Y. Kohara for sharing cDNA clones; and G. Thio for providing genomic
DNA from daf-5 mutants. We especially thank Dr M. Tewari and J. S.
Ahn for generously sharing their time to perform yeast two-hybrid assays. Some
strains in this work were provided by the C. elegans Genetics Center, which is
supported by the National Center for Research Resources. This work was
supported by NIH grant GM60994.

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